Volume 23, Issue 7, Pages 875-882 (July 2016) Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo  Tsz-Leung To, Antonino Schepis, Rubén Ruiz-González, Qiang Zhang, Dan Yu, Zhiqiang Dong, Shaun R. Coughlin, Xiaokun Shu  Cell Chemical Biology  Volume 23, Issue 7, Pages 875-882 (July 2016) DOI: 10.1016/j.chembiol.2016.06.007 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Rational Design and Characterization of a ZipGFP Protease Reporter (A) Schematic diagram of ZipGFP (see Movies S1 and S2). β1–10 (in gray) is flanked with E5 and K5 (in red and orange, respectively) at the N terminus and C terminus, respectively. β11 (in blue) is flanked with K5 and E5 (in orange and red, respectively) at the N terminus and C terminus, respectively. The protease cleavage sequence is shown in pink. The black dashed line represents an amino acid linker. The green color indicates restored green fluorescence. (B) The green fluorescence normalized by co-expressed mCherry for unmodified (i.e., unzipped) or modified (i.e., zipped) β1–10 and β11 as in (A). The error bars are standard deviations based on analysis of ∼50 cells. (C) Fluorescence images of ZipGFP with the TEV cleavage sequence expressed in HEK293 cells. Scale bar, 10 μm. (D) Emission spectra of ZipGFP over time. The two parts of cleaved ZipGFP were purified and mixed (β1–10, 2.7 μM; β11, 6.3 μM). The fluorescence spectra were then taken every 6 min. (E) The green fluorescence intensity over time after mixing the two cleaved parts of ZipGFP as in (D). The fluorescence intensity was calculated based on the emission spectra by integrating emission from 490 to 550 nm. Cell Chemical Biology 2016 23, 875-882DOI: (10.1016/j.chembiol.2016.06.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Characterization and Comparison of ZipGFP Caspase Reporter in Mammalian Cells (A) Time-lapse confocal images of human glioblastoma cells LN229 expressing ZipGFP caspase reporter upon treatment with 1 μM staurosporine (see Movie S5). mCherry was co-expressed. (B) Confocal image of the LN229 cell expressing ZipGFP caspase reporter and stained with caspase imaging agent FLICA after addition of staurosporine. Left, green fluorescence from GFP; middle, red fluorescence from co-expressed mCherry; right, far-red fluorescence from FLICA 660 caspase-3/-7 reagent. (C) Normalized green fluorescence of LN229 cells expressing ZipGFP caspase reporter reveals 5- to 10-fold fluorescence increase and cell-to-cell heterogeneity of caspase activation upon addition of staurosporine. The green fluorescence was normalized by co-expressed mCherry red fluorescence based on confocal images of live cells, which were collected every 10 min, after treatment with 1 μM staurosporine. (D) Normalized green fluorescence of LN229 cells expressing CA-GFP caspase reporter reveals <1-fold fluorescence increase upon addition of staurosporine. mLumin, a red fluorescent protein, was co-expressed and used to normalize the CA-GFP fluorescence. (E) Normalized green fluorescence of LN229 cells expressing VC3AI caspase reporter reveals 2- to 6-fold fluorescence increase upon addition of staurosporine. The red fluorescent protein mCherry was co-expressed and used to normalize the VC3AI fluorescence. Scale bars, 20 μm (A); 15 μm (B). Cell Chemical Biology 2016 23, 875-882DOI: (10.1016/j.chembiol.2016.06.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 ZipGFP Caspase Reporter in Imaging Apoptosis in Zebrafish (A) Confocal images of the zebrafish embryo at the 50% epiboly stage expressing ZipGFP caspase reporter and mCherry (see Movie S7). (B) Time-lapse fluorescence images of zebrafish embryo in the zoomed area in (A) after addition of cycloheximide. (C) Confocal images of the control zebrafish embryo (no addition of cycloheximide) expressing ZipGFP caspase reporter at 12 hpf. Arrow points to one of the somites. (D) Time-lapse images of the brain of the zebrafish embryo expressing ZipGFP caspase reporter and mCherry. Imaging started at 24 hpf (see Movies S8, S9, and S10). Fb, forebrain; R, retina; L, lens. The dashed lines outline the retina. (E) Images around the trunk of the zebrafish embryo expressing ZipGFP caspase reporter and mCherry at 48 hpf. Arrow points to the tail bud. Scale bars, 200 μm (A); 50 μm (B); 200 μm (C); 80 μm (D and E). Cell Chemical Biology 2016 23, 875-882DOI: (10.1016/j.chembiol.2016.06.007) Copyright © 2016 Elsevier Ltd Terms and Conditions